Ketones and aldehydes are polar chemical functionalities having a carbonyl group linked to one or two other carbon atoms. Ketone and aldehydes compounds play an important role in industry, agriculture, and medicine. Ketones and aldehydes are also important agents in human metabolism and biochemistry. Ketosteroids, in particular, are a class of ketone-containing steroid compounds and are uniquely valuable in research and clinical diagnosis because these compounds are critical agents in hormone-regulated biological processes and have strong biological activity at very low concentrations. Many ketosteroids are also potentially valuable pharmaceutical agents and the analysis of their function and metabolism in the body are useful in both medical treatments and diagnostic techniques for the detection of disease.
Analysis and measurement of ketone and aldehyde compounds is challenging because these compounds can be present at low levels in clinical and biological samples such as plasma. Standard chromatographic techniques such as GC-MS methods for analysis after chemical derivatization are available but the chemical derivatization is not specific. See, e.g., Song, J. et al., Journal of Chromatography B., Vol. 791, Issues 1-2, (127-135) 2003. Methods using fluorescence detection are available and some specific immunoassays, including radioimmunoassays (RAs), are available, but these usually do not offer multi-component analysis. The major problems with RAs are lack of specificity and the need to perform a different assay for each steroid.
Examples in the literature of LC/MS strategies exploit derivatization of the analytes, but the ionization efficiency is relatively low and these strategies have failed to achieve the limit of detection required for the assay to be viable in a clinical setting using mass spectrometry and also lack multiplexing capability. Steroid analysis in biological samples is also crucial for the evaluation and clinical detection of various endocrine and metabolic disorders. Clinical laboratories are currently performing radioimmunoassays (RAs) for high throughput screening of steroids.
The above challenges posed by attempting to measure ketone and aldehyde compounds in samples are also magnified by the desire to rapidly screen and/or analyze a large number of biological samples for the specific compounds of interest or a panel of ketone or aldehyde analytes. Although mass spectrometry can provide rapid throughput, ketone and aldehyde steroids are particularly challenging because of interference in the mass measurements by competing compounds and low sample concentrations in the sample medium. In addition, some classes of ketone and aldehyde compounds, and particularly ketosteroids, are not compatible with traditional sample processing conditions often used to prepare samples for mass spectrometric analysis. Ketosteroids are also particularly challenging due to poor ionization efficiency and complex ionization patterns during MS/MS analysis.
Therefore, although techniques for rapid and efficient analysis and quantitation of ketone and aldehyde compounds are highly desirable because of the biological importance of these compounds, the existing techniques are not ideal due to lack of sensitivity, cross-reacting substances, and other challenges inherent in the chemistry of the compounds.
Sensitive, selective, and accurate analysis of ketosteriods can be used for the monitoring of abnormal adrenal functions. The ionization efficiency of native ketosteriods in positive MS/MS can be poor, resulting often times in insufficient limits of detection (LODs), especially when analyzing human samples from infants and children. Derivatization of ketosteroids via their keto functionality to form hydrazines has been used to improve ionization and enhance sensitivity, as described, for example, in Kushnir et al., Performance Characteristics of a Novel Tandem Mass Spectrometry Assay For Serum Testosterone, Clin Chem. 52:1, 120-128, 2006, which is incorporated herein in its entirety by reference.
MRM analysis and MS/MS conditions work well in clean solvent, however, when using complex biological samples, a high background (BKG) noise, often from the same mass Q1/Q3 interfaces, is produced, complicating chromatography and reducing detection limits. A need exits for a method to quantitate ketosteroids and analytes containing a keto or aldehyde functionality.